Nuclease, deoxyribo-

Endpoint summary

Description of key information

13-week oral toxicity: The no-observed-adverse-effect level (NOAEL) was considered to be 100% of the Deoxyribonuclease batch PPW70965, 1012.8 mg enzyme concentrate dry matter/kg bw/day. 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 February 2016 to 29 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

revised 3 October 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

RccHan™:WIST rat

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS Limited.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 41 to 47 days.
- Weight at study initiation: Males: 139 to 182 g, Females: 116 to 141 g
- Housing: Polycarbonate cage with a stainless steel mesh lid and wood based bedding, changed at appropriate intervals. It also contained Aspen chew block and plastic shelter.
- Diet: Teklad 2014C pelleted diet non-restricted (however, removed overnight before blood sampling for hematology or blood chemistry).
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals (non-restricted).
- Acclimation period: 12 days before commencement of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24ºC
- Humidity (%): 40-70%
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light: 12 hours dark.

IN-LIFE DATES: 22 February 2016 to 21 March 2016

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

reverse osmosis water

Details on oral exposure:

DOSE VOLUME APPLIED: 10 mL/kg body weight.

DOSAGE PREPARATION: The test enzyme was provided deep frozen in a number of containers that were divided further into aliquots for each day of treatment.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose samples were analysed according to GLP.
Stability of formulations at 10 and 100%, when retained for up to 24 hours at refrigerated (nominally 5°C) or ambient temperature was demonstrated by Novozymes A/S. Samples of each formulation prepared for administration in Week 1 of treatment were analyzed for achieved concentration of the test item. Six samples of each dose concentration, taken from the middle of formulations prepared for dosing during treatment Week 1, were despatched to the Principal Investigator for analysis with regard to total nitrogen content (N-total %). Results from this analysis demonstrated acceptable formulation.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Dose / conc.:

118.9 other: enzyme concentrate dry matter/kg BW/day

Dose / conc.:

390.8 other: enzyme concentrate dry matter/kg BW/day

Dose / conc.:

1 184.1 other: enzyme concentrate dry matter/kg BW/day

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for the selection of the starting dose: The doses in this study were selected based on results from a seven-day preliminary toxicity study. In this preliminary study, the highest dose, 100% of Deoxyribonuclease, batch PPW38822, was well-tolerated and was thus selected as the top dose for the four-week study.

Positive control:

Not included

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each animal was recorded one week before treatment commenced, on the day that treatment commenced (Day 0), weekly thereafter, Day 28 (before overnight food withdrawal before blood sampling for hematology and blood chemistry) and on the day of necropsy.

FOOD CONSUMPTION:
- The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started and for each week throughout the study

WATER CONSUMPTION:
- Time schedule for examinations: Fluid intake was assessed by daily visual observation.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected after overnight withdrawal of food and prior to dosing (where appropriate) on Day 29 (at termination)
- Anaesthetic used for blood collection: Yes (isoflurane anaesthesia)
- Animals fasted: Yes, overnight
- How many animals: From all animals
- Parameters checked: Haematocrit (Hct), Haemoglobin (Hb), Erythrocyte count (RBC), Mean cell haemoglobin (MCH), Mean cell haemoglobin concentration (MCHC), Mean cell volume (MCV), Total white cell count (WBC), Platelet count (Plt), Prothrombim time (PT), Activated partial thromboplastin time (APTT)

Differential WBC count:
Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected after overnight withdrawal of food and prior to dosing (where appropriate) on Day 29 (at termination)
- Animals fasted: Yes, overnight
- How many animals: From all animals
- Parameters checked:
Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Urea, Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Triglycerides (Trig), Sodium (Na), Potassium (K), Total protein (Total Prot), Albumin (Alb), Albumin/globulin ratio (A/G Ratio)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Sensory reactivity and grip strength assessments were performed (before dosing) on all animals during Week 4 of treatment.
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity: Yes

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Other examinations:

FAECAL ANALYSIS: No

Hematology, Bone Marrow: Yes

Weight of individual organs: Yes
- Time schedule for collection of organs: At necropsy

Statistics:

Significant differences between the groups compared were expressed at the 5% (p<0.05) or 1% (p<0.01) level. The most common tests used were:
L: Data were log transformed for the statistical analysis
Du: Treated groups compared with Control using Dunnett’s test
Fe: Treated groups compared with Control using Fisher’s Exact test
Sh: Treated groups compared with Control using Shirley’s test
Wi :Treated groups compared with Control using Williams’ test

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Males receiving 100% of Deoxyribonuclease, batch PPW38822 a minor reduction of group mean body weight gain was observed throughout the treatment period when compared with controls, however, the overall mean body weight gain for these males did not attain statistical significance. Females were unaffected by treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The biochemical evaluation of the blood plasma after four weeks of treatment indicated, when compared with the controls, a minor decrease in albumin concentration, with a consequentially low albumin to globulin ratio, in females receiving 33 or 100% Deoxyribonuclease, batch PPW38822 but all individual values were within background control data ranges, consequently this was not considered to be an adverse response to treatment. In males treated at 100% of Deoxyribonuclease, batch PPW38822, a minor increase of creatinine concentration was also observed when compared with controls; although the majority of the creatinine values for males treated at 100% of Deoxyribonuclease, batch PPW38822 were within the Control range.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

After four weeks of treatment the females given 33 or 100% Deoxyribonuclease, batch PPW38822 had slightly higher adjusted kidney weights than the controls, attaining statistical significance in the animals given 100% Deoxyribonuclease, batch PPW38822. However, the values for 4/5 females, were within historical control data ranges and there was no similar finding in males. All other inter-group differences from controls were minor, lacked dose-relationship or were confined to one sex and were therefore attributed to normal biological variation.

Gross pathological findings:

no effects observed

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Motor activity during the first six-minutes of the one-hour assessment period was high, compared to the controls, in males receiving 100% of the Deoxyribonuclease, batch PPW38822 and tended to remain slightly high throughout the remainder of this
assessment period in respect of the high beam scores, and up to approximately 30 minutes in respect of the low beam scores. As a consequence, the total high and low beam scores for these animals were statistically significantly higher than those of the controls.

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 184.1 mg/kg bw/day (nominal)

Based on:

other: enzyme concentrate dry matter

Sex:

male/female

Basis for effect level:

other: No adverse effects were seen so NOAEL was the highest dose administered.

Critical effects observed:

no

Conclusions:

It was concluded that oral administration of deoxyribonuclease, batch PPW38822 to Han Wistar rats revealed a no-observed adverse-effect level (NOAEL) of 1184.1 mg enzyme concentrate dry matter/kg BW/day, which was the highest dose administered.

Executive summary:

The objective of this study was to assess the systemic toxic potential of deoxyribonuclease, batch PPW38822, when administered orally by gavage to Han Wistar rats for four weeks. Three groups, each comprising five males and five females, received doses of 10, 33 or 100% of deoxyribonuclease, batch PPW38822 (equivalent to 118.9, 390.8 or 1184.1 mg enzyme concentrate dry matter/kg/day). A similarly constituted group received the vehicle (reverse osmosis water) at the same volume-dose (10 mL/kg body weight).

During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, water consumption (by visual assessment), hematology (peripheral blood), blood chemistry, organ weight, macropathology and histopathology investigations were undertaken.

General appearance, water consumption, behavior, sensory reactivity responses and grip strength were not affected by treatment and there were no deaths during the treatment period.

Motor activity was increased in Week 4 in males receiving 100% of deoxyribonuclease, batch PPW38822. There was no adverse effect of treatment on body weight gain or food consumption although at 100% of deoxyribonuclease, batch PPW38822 it was observed that there was a minor reduction of weight gain in males throughout the treatment period; overall body weight gain did not attain statistical significance. Biochemical evaluation of blood plasma after four weeks of treatment indicated slightly low albumin concentration, with a consequentially low albumin to globulin ratio, in females receiving 33 or 100% of deoxyribonuclease, batch PPW38822, and a minor increase of plasma creatinine concentration in males receiving 100% of deoxyribonuclease, batch PPW38822. Kidney weights were slightly high after four weeks of treatment in females given 33 or 100% of deoxyribonuclease, batch PPW38822; males were unaffected. There were no treatment-related macroscopic or histopathological findings.

It was concluded that oral administration of deoxyribonuclease, Batch PPW38822, to Han Wistar rats at doses up to 100% of the deoxyribonuclease batch for four weeks caused minor non-adverse fluctuations of body weight in males and kidney weight in females. Consequently, the no-observed adverse-effect level (NOAEL) was considered to be 100% of the deoxyribonuclease batch (equivalent to 1184.1 mg enzyme concentrate dry matter/kg/day).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 006 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Data waiving:

exposure considerations

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Data waiving:

exposure considerations

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The repeated dose inhalation and dermal toxicity studies were waived.

- The dermal study was waived because of the low likelihood of absorption of enzymes through the skin due to the physico-chemical properties of the enzyme protein.

- The inhalation study was waived because exposure is too low to exert any toxicity. Potential exposure by inhalation to an amount of enzyme, which is toxicologically relevant, is unrealistic due to the stringent work practices and the formulation of enzymes, enforced because of the risk of sensitisation by inhalation.

Justification for classification or non-classification

Based on repeated dose oral study and weight of evidence, deoxyribonuclease does not exert any repeated dose oral, dermal or inhalation toxicity to workers or consumers.